Oxidative stress plays a critical role in the development of vascular disease (Barañano D. E. et al. (2002) “BILIVERDIN REDUCTASE: A MAJOR PHYSIOLOGIC CYTOPROTECTANT,” Proc. Natl. Acad. Sci (USA) 99(25):16093-16098). Cells use multiple systems to protect against reactive oxygen species. The concentration of free heavy metals, which can catalyze the formation of free radicals, are tightly regulated by chelators such as ferritin and transferrin. Enzymes with antioxidant actions include catalase and superoxide dismutase, which together convert superoxide radicals into water. Small molecules, such as ascorbate and α-tocopherol act as direct antioxidants, quenching the propagation of free radicals. Glutathione occurs at millimolar concentrations in most tissues and is generally regarded as the principal endogenous intracellular small molecule antioxidant cytoprotectant.
Bilirubin is a lipophilic linear tetrapyrrole that occurs uniquely in mammals, and is abundant in plasma (Beri, R. et al. (1993) “CHEMISTRY AND BIOLOGY OF HEME. EFFECT OF METAL SALTS, ORGANOMETALS, AND METALLOPORPHYRINS ON HEME SYNTHESIS AND CATABOLISM, WITH SPECIAL REFERENCE TO CLINICAL IMPLICATIONS AND INTERACTIONS WITH CYTOCHROME P-450,” Drug Metab Rev. 25(1-2):49-152; Yamamoto, T. (1968) “SYNTHESIS OF BILIRUBIN,” Naika Hokan. 15(11):391-398; Moore, M. R. (1980) “THE BIOCHEMISTRY OF THE PORPHYRINS,” Clin Haematol. 9(2):227-252). In humans, approximately 250-400 mg of bilirubin are formed daily as the final metabolic product of heme catabolism, as heme oxygenase (HO) cleaves the heme ring to form biliverdin (Yoshida, T. et al. (2000) “MECHANISM OF HEME DEGRADATION BY HEME OXYGENASE,” J. Inorg. Biochem. 82(1-4):33-41; Montellano, P. R. (2000) “THE MECHANISM OF HEME OXYGENASE,” Curr Opin Chem Biol. 4(2):221-227; Galbraith R. (1999) “HEME OXYGENASE: WHO NEEDS IT?,” Proc Soc Exp Biol Med. 222(3):299-305), which is then reduced by biliverdin reductase (BVR) to yield bilirubin (Wilks, A. (2002) “HEME OXYGENASE: EVOLUTION, STRUCTURE, AND MECHANISM,” Antioxid. Redox Signal. 4(4):603-614; Mantle, T. J. (2002) “H AEM DEGRADATION IN ANIMALS AND PLANTS,” Biochem Soc Trans. 30(4):630-633; Ogawa, K. (2002) “HEME METABOLISM IN STRESS RESPONSE,” Nippon Eiseigaku Zasshi 56(4):615-21). Approximately 80% of serum bilirubin is derived from hemoglobin of senescent erythrocytes that have been phagocytized by macrophages in the reticuloendothelial system (Shibahara, S. et al. (2002) “HEME DEGRADATION AND HUMAN DISEASE: DIVERSITY IS THE SOUL OF LIFE,” Antioxid Redox Signal. 4(4):593-602); the remainder derives from the catabolism of other haemoproteins and from the destruction of maturing red blood cells in the marrow.
Bilirubin has been found to possesses strong antioxidant potential against peroxyl and other reactive oxygen radicals (McGeary, R. P. et al. (2003) “BIOLOGICAL PROPERTIES AND THERAPEUTIC POTENTIAL OF BILIRUBIN,” Mini Rev Med Chem. 3(3):253-256; Stocker, R. et al. (1987) “BILIRUBIN IS AN ANTIOXIDANT OF POSSIBLE PHYSIOLOGICAL IMPORTANCE,” Science 235, 1043-1046; Hidalgo, F. J. et al. (1990) “CAN SERUM BILIRUBIN BE AN INDEX OF IN VIVO OXIDATIVE STRESS?,” Med Hypotheses 33(3):207-211; Stocker, R. et al. (1987) “ANTIOXIDANT ACTIVITY OF ALBUMIN-BOUND BILIRUBIN,” Proc. Natl. Acad. Sci. USA 84:5918-5922; Machlin, L. J. et al. (1987) “FREE RADICAL TISSUE DAMAGE: PROTECTIVE ROLE OF ANTIOXIDANT NUTRIENTS,” FASEB J. 1(6):441-445; Otterbein, L. E. et al. (2003) “HEME OXYGENASE-1: UNLEASHING THE PROTECTIVE PROPERTIES OF HEME,” Trends Immunol. 24(8):449-455; Wang, H. D. et al. (2002) “BILIRUBIN AMELIORATES BLEOMYCIN-INDUCED PULMONARY FIBROSIS IN RATS,” Am J Respir Crit Care Med. 165(3):406-411).
Several epidemiological studies have found that bilirubin levels are inversely associated with coronary artery disease and mortality from myocardial infarction (Scriver, C. R. (1995) “THE METABOLIC AND MOLECULAR BASES OF INHERITED DISEASE,” (McGraw-Hill, New York; Vitek, L. et al. (2002) “GILBERT SYNDROME AND ISCHEMIC HEART DISEASE: A PROTECTIVE EFFECT OF ELEVATED BILIRUBIN LEVELS,” Atherosclerosis 160:449-456; Schwertner, H. A. et al. (1994) “ASSOCIATION OF LOW SERUM CONCENTRATION OF BILIRUBIN WITH INCREASED RISK OF CORONARY ARTERY DISEASE,” Clin. Chem. 40:18-23; Hopkins, P. N. et al. (1996) “HIGHER SERUM BILIRUBIN IS ASSOCIATED WITH DECREASED RISK FOR EARLY FAMILIAL CORONARY ARTERY DISEASE,” Arterioscler. Thromb. Vasc. Biol. 16:250-255; Djousse, L. et al. (2001) “TOTAL SERUM BILIRUBIN AND RISK OF CARDIOVASCULAR DISEASE IN THE FRAMINGHAM OFFSPRING STUDY,” Am. J. Cardiol. 87:1196-200; Heyman, E. et al. (1989) “RETINOPATHY OF PREMATURITY AND BILIRUBIN,” N. Engl. J. Med. 320:256; Temme, E. H. et al. “SERUM BILIRUBIN AND 10-YEAR MORTALITY RISK IN A BELGIAN POPULATION,” (2001) Cancer Causes Control 12:887-894).
The possibility that the administration of bilirubin might find utility in providing cytoprotection is, however, encumbered by the toxicity and insolubility of the molecule (Hansen, T. W. (2002) “MECHANISMS OF BILIRUBIN TOXICITY: CLINICAL IMPLICATIONS,” Clin Perinatol. 29(4):765-778; Wennberg, R. P. (1991) “CELLULAR BASIS OF BILIRUBIN TOXICITY,” N Y State J Med. 91(11):493-496; Bratlid, D. (1991) “BilIRUBIN TOXICITY: PATHOPHYSIOLOGY AND ASSESSMENT OF RISK FACTORS,” N Y State J Med. 91(11):489-492). Excessive elevations of bilirubin lead to substantial deposits in the brain with the resultant kernicterus causing major brain damage (Barañano D. E. et al. (2002) “BILIVERDIN REDUCTASE: A MAJOR PHYSIOLOGIC CYTOPROTECTANT,” Proc. Natl. Acad. Sci (USA) 99(25):16093-16098; Orth, J. (1875) Virchows Arch. Pathol. Anat. 63, 447-462).
In contrast to bilirubin, biliverdin is soluble. It can be produced by incubating bilirubin in the presence of a bilirubin oxidase (E.C.1.3.3.5). A number of enzymes with bilirubin oxidase activity from various plant sources are known (See, U.S. Pat. Nos. 5,624,811; 4,985,360 and 4,770,997, European Patent Documents Nos. EP 0 140 004; EP 0 247 846; EP 0 005 637 and EP 0 320 095, and German Patent Document No. DE 32 39 236).
Biliverdin has been proposed to be potentially useful as a cytoprotective therapeutic agent (Barañano D. E. et al. (2002) “BILIVERDIN REDUCTASE: A MAJOR PHYSIOLOGIC CYTOPROTECTANT,” Proc. Natl. Acad. Sci (USA) 99(25):16093-16098; Colpaert, E. et al. (2002) “INVESTIGATION OF THE POTENTIAL MODULATORY EFFECT OF BILIVERDIN, CARBON MONOXIDE AND BILIRUBIN ON NITRERGIC NEUROTRANSMISSION IN THE PIG GASTRIC FUNDUS,” Eur. J. Pharmacol. 457(2-3):177-86; Nakagami, T. et al. (1992) “ANTIVIRAL ACTIVITY OF A BILE PIGMENT, BILIVERDIN, AGAINST HUMAN HERPESVIRUS 6 (HHV-6) IN VITRO,” Microbiol Immunol 36(4):381-390; Katori, M. et al. (2002) “A NOVEL STRATEGY AGAINST ISCHEMIA AND REPERFUSION INJURY: CYTOPROTECTION WITH HEME OXYGENASE SYSTEM,” Transpl Immunol. 9(2-4):227-233; Ryter, S. W. et al. (2000) “THE HEME SYNTHESIS AND DEGRADATION PATHWAYS: ROLE IN OXIDANT SENSITIVITY. HEME OXYGENASE HAS BOTH PRO- AND ANTIOXIDANT PROPERTIES,” Free Radic Biol Med 28(2):289-309; U.S. Patent Application Publication No. 20030162826).
In particular, biliverdin has been proposed to be useful to treat vasoconstriction (U.S. Patent Application Publication No. 20030027124), coronary artery disease (Vitek, L. et al. (2002) “GILBERT SYNDROME AND ISCHEMIC HEART DISEASE: A PROTECTIVE EFFECT OF ELEVATED BILIRUBIN LEVELS,” Atherosclerosis 160:449-456; Schwertner, H. A. et al. (1994) “ASSOCIATION OF LOW SERUM CONCENTRATION OF BILIRUBIN WITH INCREASED RISK OF CORONARY ARTERY DISEASE,” Clin. Chem. 40:18-23; Hopkins, P. N. et al. (1996) “HIGHER SERUM BILIRUBIN IS ASSOCIATED WITH DECREASED RISK FOR EARLY FAMILIAL CORONARY ARTERY DISEASE,” Arterioscler. Thromb. Vasc. Biol. 16:250-255; Djousse, L. et al. (2001) “TOTAL SERUM BILIRUBIN AND RISK OF CARDIOVASCULAR DISEASE IN THE FRAMINGHAM OFFSPRING STUDY,” Am. J. Cardiol. 87:1196-200; Heyman, E. et al. (1989) “RETINOPATHY OF PREMATURITY AND BILIRUBIN,” N. Engl. J. Med. 320:256; Temme, E. H. et al. “SERUM BILIRUBIN AND 10-YEAR MORTALITY RISK IN A BELGIAN POPULATION,” (2001) Cancer Causes Control 12:887-894) and ischemia/reperftision injury (Fondevila, C. (2003) “BILIVERDIN PROTECTS RAT LIVERS FROM ISCHEMIA/REPERFUSION INJURY,” Transplant Proc. 35(5):1798-1799). Biliverdin has been found to block acetaminophen-induced injury (Chiu, H. et al. (2002) “DIFFERENTIAL INDUCTION OF HEME OXYGENASE-1 IN MACROPHAGES AND HEPATOCYTES DURING ACETAMINOPHEN-INDUCED HEPATOTOXICITY IN THE RAT: EFFECTS OF HEMIN AND BILIVERDIN,” Toxicol Appl. Pharmacol. 181(2):106-115), and liver graft injury (Kato, Y. et al. (2003) “BILIRUBIN RINSE: A SIMPLE PROTECTANT AGAINST THE RAT LIVER GRAFT INJURY MIMICKING HEME OXYGENASE-1 PRECONDITIONING,” Hepatology 38:364-373).
Despite improved methods of producing biliverdin, a need continues to exist for efficient and inexpensive methods for producing biliverdin, and in particular, methods capable of producing a single biliverdin isomer. The present invention is directed to such needs.